Hydrolyzed collagen compositions and methods of making thereof

ABSTRACT

Disclosed herein are hydrolyzed collagen compositions. The compositions are inexpensive to make and can be produced without the use of proteolytic enzymes, decolorizing agents, antibacterial and antifungal agents, and the like. Further, the compositions are substantially free of odors and are white to light yellow in color and are suitable to be used as dietary supplements. Also disclosed are methods for producing the compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority upon U.S. provisional application Ser.No. 62/311,575 filed Mar. 22, 2016. This application is herebyincorporated by reference in its entirety for all of its teachings.

BACKGROUND

Collagen preparations have been used in a variety of capacities, fromgelling agents in food, to pharmaceuticals and health aids, tophotography, cosmetic manufacturing, and adhesives. Within the healthindustry, collagen preparations have been used to enhance athleticperformance by supplying essential amino acids and shortening recoverytime after exercise, and have also been used as weight loss aids anddigestive aids as well as to relieve pain, reduce inflammation, andimprove joint function for patients with osteoarthritis and rheumatoidarthritis. Collagen preparations have been used as supplements to combatosteoporosis and improve the condition of fingernails and hair. In thecosmetic industry, meanwhile, collagen preparations are available astopical products for use on the skin to increase skin suppleness, reducethe appearance of wrinkles and fine lines, and to reduce the appearanceof stretch marks.

Although the raw materials for extracting collagen are readily availableas byproducts from other industries, such as the fishing and poultryprocessing industries, current methods for producing collagen areexpensive and time-consuming Cartilage tissue must be separated frommeat and bones and chopped. Hydrolysis of collagen often requires thesequential use of multiple enzymes, each of which likely performs bestin a different set of reaction conditions (e.g., temperature, saltcontent, solution pH, etc.). Further, specialized digestive enzymes areexpensive and the cost increases when multi-enzyme cocktails are used,especially on an industrial scale. Following hydrolysis, a number ofpurification steps are often required, including the use of decolorizingagents, antibacterial and antifungal agents, and the like. Product odoris also a consideration; collagen preparations with strong odors will beunsuitable especially for incorporation into cosmetic or food products.

It would thus be advantageous to have an enzyme-free process forproducing hydrolyzed collagen preparations. It would further beadvantageous if this process resulted in a product or products with awhite or light yellow color and minimal to no odor. Still further, itwould be advantageous if collagen preparations produced according tothis process were able to supply essential minerals in addition to theamino acids and peptides naturally present in the preparations. Thepresent invention addresses these needs.

SUMMARY

Disclosed herein are hydrolyzed collagen compositions. The compositionsare inexpensive to make and can be produced without the use ofproteolytic enzymes, decolorizing agents, antibacterial and antifungalagents, and the like. Further, the compositions are substantially freeof odors and are white to light yellow in color and are suitable to beused as dietary supplements. Also disclosed are methods for producingthe compositions.

The advantages of the invention will be set forth in part in thedescription that follows, and in part will be obvious from thedescription, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

DETAILED DESCRIPTION

Before the present compounds, compositions, and/or methods are disclosedand described, it is to be understood that the aspects described beloware not limited to specific compounds, synthetic methods, or uses, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an acid” includes mixtures of two or more such acids, andthe like.

“Optional” or “optionally” means that the subsequently-described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

As used herein, “cartilage” refers to a type of connective tissue inanimals It is a flexible tissue that is somewhat more rigid than muscleand is found in various locations throughout the body including joints,the rib cage, the nose and ear, bronchial tubes, trachea, between thevertebra, and so forth. Cartilage tissue contains a large amount ofextracellular matrix that is high in collagen and other proteoglycans.Chondrocytes are matrix-producing cells that have become trapped in thematrix.

“Collagen” is a structural protein found in connective tissue; itfrequently takes the form of fibrils arranged in a triple helix.Fibrillar types of collagen include Types I, II, III, V, and XI. Type Icollagen makes up a great deal of the organic part of bone as well asbeing found in skin, tendons, blood vessels, and organs, while type IIIcollagen is commonly found near or with type I. On the other hand,cartilage is composed primarily of type II collagen. Other types ofcartilage are less common and may be found in membranes, on cellsurfaces, and associated with hair and placental structures.

As used herein, “hydrolysis” refers to the breaking of a chemical bondin a molecule via the molecule's reaction with water. Thus, a nucleicacid would be hydrolyzed into oligonucleotides and componentnucleotides, a protein into peptides and amino acids, a polysaccharideinto oligosaccharides and component sugars, and so forth. The degree ofcompletion of hydrolysis is dependent upon reaction conditions.Hydrolysis can be accomplished in basic solution, in acidic solution, ina salt solution (usually when the salt is a weak acid or base), or usingan enzyme.

“Hydrolyzed collagen” as used herein is collagen that has been subjectedto a hydrolysis process. “Gelatin” is collagen that has been partiallyhydrolyzed and possesses specific properties such as solubility in hotwater and a similar amino acid composition to the parent collagen.

The sternum, or breastbone, is a large bone to which the pectoralmuscles are attached. In avians such as chickens, the sternum ispositioned under the body and is enlarged in size for attachment ofpowerful flight muscles. Avian sterna are typically associated with alarge proportion of cartilage that is rich in type II collagen.

As used herein, “neutralization” refers to the treatment of an acidicreaction solution with an approximately quantitative amount of a base.Neutralization with base can stop an acidic hydrolysis reaction, forexample.

An “anti-bacterial agent” is any compound or composition or treatmentthat destroys bacteria. Alternatively, an anti-bacterial agent cansuppress the growth of bacteria or can prevent bacteria fromreproducing. Ultraviolet light, heat treatment, certain chemicals suchas bleach or ethanol, and antibiotics are considered anti-bacterial.

As used herein, the term “about” is used to provide flexibility to anumerical range endpoint by providing that a given value may be “alittle above” or “a little below” the endpoint without affecting thedesired result.

Concentrations, amounts, and other numerical data may be expressed orpresented herein in a range format. It is to be understood that such arange format is used merely for convenience and brevity and thus shouldbe interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range, but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited. Asan illustration, a numerical range of “about 1 to about 5” should beinterpreted to include not only the explicitly recited values of about 1to about 5, but also to include individual values and sub-ranges withinthe indicated range. Thus, included in this numerical range areindividual values such as 2, 3, and 4 and sub-ranges such as from 1-3,from 2-4, and from 3-5, etc., as well as 1, 2, 3, 4, and 5,individually. This same principle applies to ranges reciting only onenumerical value as a minimum or a maximum. Furthermore, such aninterpretation should apply regardless of the breadth of the range orthe characteristics being described.

As used herein, a plurality of items, structural elements, compositionalelements, and/or materials may be presented in a common list forconvenience. However, these lists should be construed as though eachmember of the list is individually identified as a separate and uniquemember. Thus, no individual member of such list should be construed as ade facto equivalent of any other member of the list solely based ontheir presentation in a common group without indications to thecontrary.

Disclosed are materials and components that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed compositions and methods. These and other materials aredisclosed herein, and it is understood that when combinations, subsets,interactions, groups, etc., of these materials are disclosed, that whilespecific reference of each various individual and collective combinationand permutation of these compounds may not be explicitly disclosed, eachis specifically contemplated and described herein. For example, if anacid is disclosed and a number of different neutralizing bases arediscussed, each and every combination and permutation of acid and basethat is possible is specifically contemplated unless specificallyindicated to the contrary. For example, if a class of molecules A, B,and C are disclosed, as well as a class of molecules D, E, and F, and anexample of a combination A+D is disclosed, then even if each is notindividually recited, each is individually and collectivelycontemplated. Thus, in this example, each of the combinations A+E, A+F,B+D, B+E, B+F, C+D, C+E, and C+F, are specifically contemplated andshould be considered disclosed from disclosure of A, B, and C; D, E, andF; and the example combination of A+D. Likewise, any subset orcombination of these is also specifically contemplated and disclosed.Thus, for example, the sub-group of A+E, B+F, and C+E is specificallycontemplated and should be considered disclosed from disclosure of A, B,and C; D, E, and F; and the example combination of A+D. This conceptapplies to all aspects of this disclosure including, but not limited to,steps in methods of making and using the disclosed compositions. Thus,if there exist a variety of additional steps that can be performed withany specific embodiment or combination of embodiments of the disclosedmethods, each such combination is specifically contemplated and shouldbe considered disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component denote the weight relationshipbetween the element or component and any other elements or components inthe compound or composition for which a part by weight is expressed.Thus, in a compound containing 2 parts by weight of component X and 5parts by weight of component Y, X and Y are present at a weight ratio of2:5, and are present in such ratio regardless of whether additionalcomponents are contained in the compound.

A weight percent of a component, unless specifically stated to thecontrary, is based on the total weight of the formulation or compositionin which the component is included.

Provided herein are compositions containing collagen as well as methodsto produce the compositions. In one aspect, the collagen is hydrolyzed.In another aspect, the collagen is sourced from vertebrate cartilage. Ina further aspect, the vertebrate is a fish, mammal, or avian. In a stillfurther aspect, the vertebrate is a chicken and the cartilage is sternalcartilage, while the collagen is type II collagen.

In one aspect, provided herein is a method for producing hydrolyzedcollagen, the method including the following steps:

-   (a) heating a chicken sternum in the presence of an aqueous acid at    an elevated temperature for a time period to produce a first    composition;-   (b) filtering the first composition to remove the supernatant;-   (c) neutralizing the acid present in the supernatant to produce a    neutralized supernatant; and-   (d) removing the water in the neutralized supernatant to produce the    hydrolyzed collagen.

Each step of the process described herein is discussed in detail below.

In one aspect, prior to step (a), the chicken sternum is removed fromthe chicken skeleton. Here, all of the meat is removed from the chickensternum or substantially all of the meat is removed (e.g., greater than90%, greater than 95%, or greater than 99%).

In another aspect, the chicken sternum is treated with an anti-bacterialagent prior to step (a). Examples of anti-bacterial agents useful hereininclude, but are not limited to, ethanol, isopropanol, chlorine, bleach,a peroxide, a quaternary ammonium compound, or a combination thereof. Ina further aspect, prior to step (a), the chicken sternum is boiled thenthe temperature is reduced prior to hydrolysis. In an alternativeaspect, if the chicken sternum is processed immediately, ananti-bacterial agent is not required.

After the chicken sternum has been removed, it can be processed furtherprior to hydrolysis. For example, the chicken sternum can be chopped orpulverized to increase the surface area of the sternum.

After the chicken sternum has been removed, it is subjected to acidhydrolysis. The selection of the hydrolysis conditions in step (a) isimportant with respect to producing highly pure hydrolyzed collagen thathas little to no odor or discoloration.

In one aspect, the acid is a strong acid. In a further aspect, the acidis selected from hydrochloric acid, hydroiodic acid, hydrobromic acid,perchloric acid, nitric acid, sulfuric acid, or a combination thereof.In one aspect, the acid is present in solution at a concentration offrom 0.5 M to 4 M, or is about 0.5 M, about 1 M, about 1.5 M, about 2 M,about 2.5 M, about 3 M, about 3.5 M, or about 4 M, where any value canbe a lower and upper endpoint of a range (e.g., 1 M to 3 M). In anotheraspect, a lower acid concentration will result in less base needed forneutralization. In some aspects, when the acid concentration was lessthan 1 M, little or no hydrolysis was observed at 12 hours.

The ratio of the amount of acid to the chicken sternum is important withrespect to the purity of the final hydrolyzed collagen. In one aspect,the ratio of the chicken sternum per mole of acid is from 500 grams to5,000 grams of chicken sternum per mole of acid, or is about 500, 525,550, 575, 600, 625, 650, 675, 700, 725, 750, 1,000; 1,100; 1,200; 1,300;1,400; 1,500; 2,000; 2,500; 3,000; 3,500; 4,000; 4,500, or 5,000 gramsof chicken sternum per mole of acid, where any value can be a lower andupper endpoint of a range (e.g., 500 to 750). Thus, by varying theamount of the chicken sternum and concentration of the acid, it ispossible to vary the ratios above. The Examples provide calculations fordetermining these ratios.

The hydrolysis step (a) is conducted at an elevated temperature. In oneaspect, the elevated temperature is from 50° C. to boiling, or is about50° C., about 55° C., about 60° C., about 65° C., about 70° C., about75° C., about 80° C., about 90° C., about 95° C., or about 100° C.,where any value can be a lower and upper endpoint of a range (e.g., 55°C. to 65° C.).

The duration of the hydrolysis step (a) is also important with respectto producing highly pure hydrolyzed collagen. In one aspect, theduration of hydrolysis is from 2 hours to 48 hours, or is about 2 hours,about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours,about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about11 hours, about 11.5 hours, about 12 hours, about 24 hours, or about 48hours where any value can be a lower and upper endpoint of a range(e.g., 8 to 12 hours). In another aspect, the duration of hydrolysis isfrom 10 hours to 20 hours, 15 hours to 20 hours, 16 hours to 18 hours,or about 17 hours.

As discussed above, the conditions of the hydrolysis step are importantwith respect to producing highly pure hydrolyzed collagen. For example,hydrolysis slows when the reaction temperature drops below 50° C.Furthermore, longer heating times result in more highly-coloredproducts. In one aspect, the chicken sternum is heated in the presenceof hydrochloric acid at a temperature of from 58° C. to 62° C. for atime period of 16 to 18 hours. Further in this aspect, the chickensternum can be present in a ratio of from 500 grams to 1,000 grams permole of acid.

In another aspect, the hydrolysis reaction can be performed in open air,under vacuum, or under a nitrogen atmosphere. The hydrolysis step can beperformed in a batch process or continuous process.

In one aspect, after hydrolysis step (a), the resulting mixture isoptionally filtered to remove any insoluble materials. Standardfiltration techniques can be used in this aspect.

After the hydrolysis step (a), a neutralization step is performed. Inone aspect, the mixture produced after step (a) can be neutralizeddirectly. In another aspect, if a filtration step is performed, the acidin the supernatant is neutralized with a base. The concentration andamount of base used in the neutralization step is generally in theamount to completely neutralize all of the acid present in thesupernatant or substantially neutralize all of the acid (e.g., greaterthan 90%, greater than 95%, or greater than 99%).

In one aspect, the base is a strong base. In a further aspect, thestrong base is sodium hydroxide, potassium hydroxide, calcium hydroxide,or a combination thereof. In an alternative aspect, the base is a weakbase. Further in this aspect, the weak base is calcium carbonate, sodiumcarbonate, potassium carbonate, ammonia, sodium bicarbonate, or acombination thereof. In either of these aspects, the strong or weak basecan be an alkali metal base or an alkaline earth metal base.

In a further aspect, the acid is neutralized with calcium hydroxide orcalcium carbonate. In one aspect, calcium carbonate is preferred forneutralizing the substrate since calcium salts such as calcium chlorideare left behind in the hydrolyzed collagen composition and these aresafe for oral consumption by humans or other mammals. In a furtheraspect, the hydrolyzed collagen composition may provide some healthbenefits such as bone and joint support to a subject who consumes thecomposition. In a still further aspect, any calcium salts left behindafter drying the hydrolyzed collagen composition provide a furtherhealth benefit such as bone support to a subject who consumes thecomposition. In an alternative aspect, drying a hydrolyzed collagencomposition that has been neutralized by calcium carbonate requires lesstime and energy than drying a hydrolyzed collagen compositionneutralized with another base, since calcium carbonate adsorbs lesswater than other bases.

After neutralization, the resulting mixture can be filtered to removeany insoluble materials and isolate the supernatant containing thehydrolyzed collagen. In the alternative, if a filtration step wasperformed after hydrolysis and prior to neutralization, no additionalfiltration is required of the neutralized supernatant. The water isremoved from the neutralized supernatant to produce a dry solid orpowder of the hydrolyzed collagen. Methods for removing the water fromthe supernatant include, but are not limited to, rotary evaporation,lyophilization, oven drying, or spray drying.

Also disclosed herein are hydrolyzed collagen products produced by themethods described herein. In one aspect, the hydrolyzed collagen has anaverage molecular weight of less than 10 kDa. In a further aspect, thehydrolyzed collagen has an average molecular weight of 1 kDa, 2 kDa, 3kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, or 10 kDa, or from about3 kDa to about 5 kDa. In another aspect, the hydrolyzed collagencontains one or more calcium salts (e.g., CaCl₂) in an amount of lessthan 25% by weight of the hydrolyzed collagen, or about 5%, about 10%,about 15%, about 20%, or about 25% by weight of the hydrolyzed collagenwhere any value can be a lower and upper endpoint of a range (e.g., 10%to 25%). In one aspect, the average molecular weight of the hydrolyzedcollagen can be altered by changing the hydrolysis reaction time.

In another aspect, the hydrolyzed collagen composition described hereincontains hyaluronic acid in an amount of less than 10% by weight of thehydrolyzed collagen, or contains hyaluronic acid at about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9%by weight of the hydrolyzed collagen where any value can be a lower andupper endpoint of a range (e.g., 2% to 9%). In another aspect, thehydrolyzed collagen composition contains less than 2% by weight, lessthan 1.5% by weight, or less than 1% by weight hyaluronic acid.

In another aspect, the hydrolyzed collagen composition described hereincontains chondroitin sulfate in an amount of less than 10% by weight ofthe hydrolyzed collagen, or contains chondroitin sulfate at about 1%,about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, orabout 9% by weight of the hydrolyzed collagen where any value can be alower and upper endpoint of a range (e.g., 2% to 9%). In this aspect,the presence of chondroitin sulfate is derived from the chicken sternumand not an additional supplement. In another aspect, the hydrolyzedcollagen composition contains less than 4% by weight chondroitinsulfate. In a further aspect, hydrolyzed collagen composition containsfrom 0.5% to 4% by weight chondroitin sulfate.

In a further aspect, the hydrolyzed collagen compositions describedherein have little to no odor compared to other hydrolyzed collagencompositions. In another aspect, the hydrolyzed collagen compositionsdescribed herein have little or no color compared to other hydrolyzedcollagen compositions and appear white or light yellow in color. In oneaspect, color and odor are removed with the use of a decolorizing agentsuch as, for example, activated carbon or activated charcoal. In anotheraspect, charcoal or a decolorizing agent is not needed to remove colorand odor from the hydrolyzed collagen composition.

In one aspect, the total protein level of the hydrolyzed collagencomposition is greater than 80%, greater than 85%, or greater than 90%.In another aspect, the total collagen level of the hydrolyzed collagencomposition is greater than 60%, greater than 65%, or greater than 70%.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated. The examples are intended to be purely exemplary andare not intended to limit the scope of what the inventors regard astheir invention. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. Numerous variationsand combinations of reaction conditions, e.g., component concentrations,desired solvents, solvent mixtures, temperatures, pressures, and otherreaction ranges and conditions that can be used to optimize the productpurity and yield obtained from the described process. Only reasonableand routine experimentation will be required to optimize such processesand conditions.

Ratio of Amount of Chicken Sternum per Mole of Acid

In Example 1, the amount of cartilage treated with the HCl was 5 g per 2mL of acid. The following calculation was used to determine the amountof chicken sternum per mole of acid.

Moles of HCl in 2 mL of 2 M solution

${\frac{2\mspace{14mu} {moles}\mspace{14mu} {HCl}}{1\mspace{14mu} L} \times \frac{1\mspace{14mu} L}{1000\mspace{14mu} {mL}} \times 2\mspace{14mu} {mL}} = {0.004\mspace{14mu} {moles}\mspace{14mu} {HCl}}$

Ratio of 5 g chicken sternum treated with 2 mL of 2 M HCl (i.e., 0.004moles of HCl)

$\frac{5\mspace{14mu} g\mspace{14mu} {chicken}\mspace{14mu} {sternum}}{0.004\mspace{14mu} {moles}\mspace{14mu} {HCl}} = \frac{1,250\mspace{14mu} g\mspace{14mu} {chicken}\mspace{14mu} {sternum}}{{mole}\mspace{14mu} {HCl}}$

Thus, when 5 g of chicken sternum is treated with 2 mL of 2 M HCl, thisis equivalent to 1,250 g of chicken sternum treated with 1 mole ofaqueous HCl.

Example 1 General Procedure

The following general procedure was used to prepare collagen hydrolysatefrom chicken sternal cartilage. Some parameters were varied during thecourse of optimization of conditions (see Example 2).

(1) Sternal cartilage was removed from chicken skeleton. Cartilage waseither used immediately or stored in a volume of 70% ethanol sufficientto cover the biological material.

(2) 0.5 mL of 2M aqueous HCl per gram of cartilage was added.

(3) The mixture was placed in a reaction vessel at 60° C. and stirredvigorously for 12 hours.

(4) Optionally, the mixture was filtered. The filter was rinsed withultrapure water to remove residual soluble material.

(5) The supernatant liquid was neutralized with either 0.25 mL of sodiumhydroxide per mL of HCl originally used or, alternatively, with 0.11 gcalcium carbonate per mL of HCl originally used. Neutralization withCaCO₃ results in the evolution of carbon dioxide gas.

(6) Water is removed from the neutralized supernatant. Lyophilizationand/or spray drying were typically used. The final hydrolyzed collagenproduct is an off-white material having a “fluffy” appearance (FIGS. 1and 2).

Example 2 Experimental Permutations

Various parameters in the procedure of Example 1 were altered in orderto optimize product yield. Results from several example procedures arepresented in Table 1. Hydrolysis was evaluated using denaturing (SDS)polyacrylamide gel electrophoresis.

TABLE 1 Experimental Permutations Acid Cartilage Water Hydrolysis Sampleand (g)/Acid Ratio Temp. Time Evidence of ID Conc. (mL) (° C.) (hours)Vacuum^((f)) Base^((g)) Hydrolysis^((h)) 10 2M HCl 100  5 No 11 2M HCl90 5 No 12.1^((a)) 2M HCl 50 2 yes Yes 12.2^((a)) 2M HCl 60 2 yes Yes12.3^((a)) 2M HCl 80 2 yes Yes 13.1^((a)) 2M HCl 60 4.5 yes Yes13.2^((a)) 2M HCl 70 4.5 yes Yes 13.3^((a)) 2M HCl 80 4.5 yesinconclusive 14 2M HCl 90 8 No 14.S^((b)) 2M HCl 90 8 Yes 15 None 80 48unhydrolyzed 16 2M HCl 50 24 not tested 17 2M HCl 1:1 55-60 4.5 yes NaOHNo 18 2M HCl 1:1 50 96 no NaOH 21 2M HCl 1:1 60 4.5 no NaOH 22.1^((c))2M HCl 5:2 60-65 10 yes NaOH 22.2^((c)) 2M HCl 5:2 55-60 10 yes NaOH 232M HCl 5:2 60 10 no NaOH 24 2M HCl 5:2 60 17 no CaCO₃ 24.F^((d)) 2M HCl5:2 60 17 no CaCO₃ 24.I^((e)) 2M HCl 5:2 60 17 no CaCO₃ 25 pepsin 3% w/w35 >60 days no 26 2M HCl 7:3 60 18 no CaCO₃ ^((a))Three replicatesperformed under similar set of conditions. ^((b))Solids from filteringwere analyzed. ^((c))Two replicates performed under similar set ofconditions. ^((d))Unfiltered product analyzed. ^((e))Filtrate analyzed.^((f))Hydrolysis reaction carried out under vacuum. ^((g))Base added toneutralize acid at the completion of hydrolysis reaction.^((h))Appearance of bands in the SDS-PAGE gel that had migrated outsidethe sample wells was considered positive evidence of hydrolysis. In somecases, hydrolysis may have generated small peptides that ran off the endof the gel or were too small to stain.

Example 3 Characterization of Products

Products were subjected to HPLC analysis, method ALC190A, performed byAdvanced Botanical Consulting & Testing, Inc. (Tustin, Calif.). Thismethod detects and quantifies amino acids in bound form, after proteinhydrolysis. Results from an example product are presented in Table 2.

TABLE 2 Amino Acid Analysis Amino Acid % of Composition Histidine 0.49Arginine 3.98 Glutamine 0.12 Glycine 10.37 Serine 9.31 Glutamic acid8.92 Aspartic acid 8.87 Proline 6.87 Threonine 5.88 Alanine 0.00Tyrosine 9.67 Methionine 1.06 Lysine 3.38 Valine 0.00 Leucine 4.41Isoleucine 8.60 Phenylalanine 0.00 Taurine 4.25 Total Amino Acids 86.16(calculated)

In another experiment, hydrolysis was performed using the protocol inExample 1 with the exception that hydrolysis was conducted at 60° C. for17 hours using 1 M HCl (ratio of 5 g cartilage per 2 mL of 1 M HCl). Theamino acid content of the resulting hydrolysate is provided in Table 3(two samples).

TABLE 3 Amino Acid Analysis Amino Acid % of Composition Alanine 4.517.11 Arginine 4.66 5.35 Aspartic acid 4.52 7.58 Cystine 0.41 0.60Glutamic acid 8.19 12.36 Glycine 10.46 13.92 Histidine 1.21 1.55Isoleucine 1.61 2.49 Leucine 3.29 4.18 Lysine 2.55 4.66 Methionine 1.001.30 Phenylalanine 1.86 1.90 Proline 6.04 8.51 Serine 2.00 3.30Threonine 4.41 3.38 Tryptophan 0.29 0.34 Tyrosine 0.64 1.20 Valine 2.463.44

Throughout this publication, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the methods, compositions, and compounds herein.

Various modifications and variations can be made to the methods,compositions, and compounds described herein. Other aspects of themethods, compositions, and compounds will be apparent from considerationof the specification and practice of the methods, compositions, andcompounds disclosed herein. It is intended that the specification andexamples be considered as exemplary.

1. A method for producing hydrolyzed collagen, the method comprising (a)heating a chicken sternum in the presence of an aqueous acid at atemperature of from 55° C. to 65° C. for 8 to 20 hours, wherein theratio of chicken sternum per mole of acid is from 250 g chickensternum/mole of acid to 2,000 g chicken sternum/mole of acid to producea first composition; (b) optionally filtering the first composition toremove the supernatant; (c) neutralizing the acid present in thesupernatant to produce a neutralized supernatant; and (d) removing thewater in the neutralized supernatant to produce the hydrolyzed collagen.2. The method of claim 1, wherein chicken sternum is removed from thechicken skeleton prior to step (a).
 3. The method of claim 1, whereinchicken sternum is treated with an anti-bacterial agent prior to step(a).
 4. The method of claim 1, wherein the chicken sternum is heated inthe presence of an aqueous acid at a temperature of from 58° C. to 62°C. for 16 to 18 hours.
 5. The method of claim 1, wherein the acidcomprises hydrochloric acid.
 6. The method of claim 1, wherein the ratioof chicken sternum per mole of acid is from 500 g chicken sternum/moleof acid to 5,000 g chicken sternum/mole of acid.
 7. The method of claim1, wherein the ratio of chicken sternum per mole of acid is from 500 gchicken sternum/mole of acid to 1,000 g chicken sternum/mole of acid. 8.The method of claim 1, wherein the supernatant is neutralized with analkali metal base or an alkaline earth metal base.
 9. The method ofclaim 1, wherein the supernatant is neutralized with sodium hydroxide orsodium carbonate.
 10. The method of claim 1, wherein the supernatant isneutralized with calcium hydroxide or calcium carbonate.
 11. The methodof claim 1, wherein the water in step (d) is removed by lyophilizationor spray drying.
 12. The method of claim 1, wherein the chicken sternumis heated in the presence of aqueous hydrochloric acid at a temperatureof from 58° C. to 62° C. for 16 to 18 hours, wherein the ratio ofchicken sternum per mole of acid is from 500 g chicken sternum/mole ofacid to 1,000 g chicken sternum/mole of acid.
 13. The method of claim12, wherein the supernatant is neutralized with calcium hydroxide orcalcium carbonate.
 14. Hydrolyzed collagen produced by the method ofclaim
 1. 15. The hydrolyzed collagen of claim 14, wherein the hydrolyzedcollagen has an average molecular weight of less than 10 kDa.
 16. Thehydrolyzed collagen of claim 14, wherein the hydrolyzed collagencomprises one or more calcium salts in the amount of less than 25% byweight of the hydrolyzed collagen.
 17. The hydrolyzed collagen of claim14, wherein the hydrolyzed collagen comprises hyaluronic acid in theamount of less than 2% by weight of the hydrolyzed collagen.
 18. Thehydrolyzed collagen of claim 14, wherein the hydrolyzed collagencomprises chondroitin sulfate in the amount of less than 4% by weight ofthe hydrolyzed collagen.